Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Crude hypothalamic extracts prepared from brains of 1-day-old rats produced a dose-dependent inhibition of prolactin (Prl) release by adult male hemipituitaries, and to a lesser extent by hemipituitaries of adult ovariectomized (OVX), estrogen-primed rats. These extracts contained 6-fold lower levels of dopamine than adult hypothalami. The inhibitory effect of the adult hypothalamic extracts, contrary to infantile hypothalamic extracts could be blocked by spiroperidol. Digestion of the infantile hypothalamic extracts with pronase totally abolished their Prl release-inhibiting activity, indicating the peptidic nature of this inhibitory substance. In contrast to their effect on Prl release by hemipituitaries, infantile hypothalamic extracts stimulated Prl release from dispersed anterior pituitary cells of OVX estrogen-primed rats, pointing to the importance of estrogen in modulating prolactin release-inhibiting factor (PIF) activity and the possibility that the PIF receptor is trypsin-sensitive.
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PMID:On the presence of a nondopaminergic, peptidergic prolactin release-inhibiting factor in hypothalamic extracts of infantile rats. 287 86

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Purification and protein sequence analysis of rat liver prolactin receptor. 292 41

Previous studies have strongly, but indirectly, suggested that rat decidual tissue produces a prolactin-like hormone, decidual luteotropin, which markedly affects luteal cell function. However, it was also found that extracts of decidual tissue do not cross-react with antisera to either rat or ovine prolactin (PRL). The purpose of this study was to determine whether the decidual tissue contains a substance that binds to PRL receptors in rat luteal membranes and, if so, to identify, quantitate, and characterize this molecule with the use of an ovarian radioreceptor assay. Decidual tissue was induced in day 5 pseudopregnant rats by scratching the antimesometrial wall of the uterus; it was collected on day 9 and homogenized and extracted. Decidual tissue extracts bound specifically to ovarian PRL receptors. Graded dilutions of the extracts yielded curves that were parallel to the ovine PRL standard, indicating that decidual luteotropin competes for the same receptor sites on rat luteal membranes. To determine the levels of decidual luteotropin throughout pseudopregnancy, decidual tissue was obtained on each day between days 6-12. The PRL-like activity was detectable in decidual tissue as early as day 6, reached a maximum on day 9, and declined thereafter. The elution profile obtained from gel filtration of a day 9 decidual tissue extract displayed a major component of decidual luteotropin eluting at a Ve/Vo ratio of approximately equal to 2.0. Column chromatography indicated that decidual luteotropin corresponds to a protein with a molecular weight of 23,500. The hormone was heat labile, digestible by trypsin, and appears to contain disulfide linkages. In summary, this study reports the identification, quantitation, and partial characterization of a PRL-like hormone produced by the decidual tissue of the rat.
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PMID:Identification and partial characterization of a prolactin-like hormone produced by rat decidual tissue. 298 45

Glandular kallikrein is a major estrogen-induced protein of the rat anterior pituitary. A second kallikrein-like protease in the rat anterior pituitary (kallikrein A) is not affected by estrogens, nor is a third pituitary protease which cleaves a trypsin substrate but not kallikrein substrates. This study examined whether any of the pituitary proteases are regulated by dopaminergic mechanisms. Ovariectomized female rats were treated for 5-10 days with reserpine (a catecholamine depleting agent), haloperidol (a dopamine receptor blocker) or bromocriptine (a dopamine receptor agonist); some rats also received 1 or 2 micrograms estradiol benzoate every 48 h. Following activation of latent proteases with trypsin, anterior pituitary extracts were assayed for kallikrein activity before and after fractionation on DEAE-Sephadex to separate the two kallikrein-like proteases. Reserpine or haloperidol doubled glandular kallikrein levels in anterior pituitaries from estrogen-treated rats. Reserpine or haloperidol had little or no effect in the absence of estrogen (estrogen produced a 5- to 7-fold increase in glandular kallikrein in the absence of drug treatment). Bromocriptine markedly attenuated the ability of estrogen to induce glandular kallikrein. Further, bromocriptine blocked the ability of reserpine to increase glandular kallikrein levels, and haloperidol attenuated the effect of bromocriptine. Other anterior pituitary proteases were unaffected by either estrogen, haloperidol, reserpine or bromocriptine. The results demonstrate that the estrogen induction of glandular kallikrein in the rat anterior pituitary is modulated by inhibitory dopaminergic mechanisms. Prolactin is the only pituitary hormone which exhibits a similar profile of hormonal and neuroendocrine regulation; this suggests a possible link between glandular kallikrein and prolactin.
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PMID:Dopaminergic regulation of the estrogen-induced glandular kallikrein in the rat anterior pituitary. 302 15

In this study we evaluated the quantitative influence of GRF and TRH on the rate of hormone secretion from single cells in cultures of male pituitaries. To accomplish this, we dispersed pituitaries from male rats with trypsin and cultured them for 24 or 48 h. Reverse hemolytic plaque assays for GH and prolactin were then performed on retrypsinized cultures to identify individual cells that secreted these hormones. Mammotropes and somatotropes were found to comprise 31.4 +/- 1.8 and 32.2 +/- 0.9% (mean +/- SE, n = 3 experiments), respectively, of all cells in 24-hour cultures. Immunocytochemical staining of different batches of cells from the same dispersions corroborated the proportions of these two cell types. Differences in the rate of basal hormone secretion were observed within each of these cell populations as evidenced by the gradual appearance of prolactin and GH plaques over a 4-hour period when incubations were conducted in the absence of stimulatory secretagogues. Addition of increasing concentrations of GRF (1 X 10(-10) -1 X 10(-7) M) or TRH (1 X 10(-9) -1 X 10(-6) M) to these incubations resulted in dose-related increases in the rate of GH and prolactin plaque formation, respectively. Maximal plaque development by somatotropes could be induced within 30 min of administering large doses of GRF, indicating that most, if not all somatotropes are responsive to this secretagogue. In contrast, approximately one third of all mammotropes could not be stimulated to form plaques acutely when subjected to similar treatment with TRH. This observation suggests that mammotropes are heterogeneous with respect to TRH responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis by plaque assays of GH and prolactin release from individual cells in cultures of male pituitaries. Evidence for functional heterogeneity within rat mammotrope and somatotrope populations. 307 93

The observation that suckling evokes a modest rise in serum TSH when compared with that of prolactin is inconsistent with the hypothesis that TRH serves as a hypophysiotropic mediator of this response. In the present study we attempted to provide an explanation for this discrepancy by determining whether any of a growing number of putative prolactin releasing factors could alter pituitary responsiveness to TRH. Anterior pituitaries from lactating (day 14) rats were monodispersed with trypsin, cultured for 2 days, and then incubated in the presence of medium alone or medium containing TRH, dopamine, or a combination of these secretagogues. Companion sets of cultures were incubated concurrently with either beta-endorphin, neurotensin, oxytocin, serotonin, vasoactive intestinal polypeptide, or lysine vasopressin. As expected, TRH stimulated and dopamine suppressed prolactin release. None of the substances tested except oxytocin had a significant effect on pituitary cell responsiveness to TRH or dopamine. Oxytocin had no effect on prolactin secretion when tested alone or in combination with TRH and dopamine. TRH alone stimulated TSH release by these cultures, while oxytocin and dopamine were ineffective by themselves. However, TSH secretion by cultures treated simultaneously with TRH and oxytocin could be suppressed to approximately half of that released by cells incubated with TRH alone. These results demonstrate that oxytocin attenuates TRH-induced TSH release by a direct action on pituitary cells without affecting the prolactin response. This selectivity of responsiveness imparted by oxytocin might contribute to the blunted release of TSH after suckling.
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PMID:Oxytocin attenuates TRH-induced TSH release from rat pituitary cells. 315 75

Our studies demonstrate that rat anterior pituitary cells (GH3) are capable of synthesizing and secreting tissue kallikrein together with prolactin and growth hormone. The secretion of prolactin and growth hormone in GH3 cells was measured by two newly developed sensitive radioimmunoassays (RIA), using the polyethylene glycol separation technique. In the direct radioimmunoassay for rat tissue kallikrein, using a polyclonal antiserum which recognizes both active and prokallikrein, the GH3 kallikrein displays parallelism with standard curves of rat urinary kallikrein. The production of immunoreactive kallikrein, prolactin, and growth hormone is time-dependent, and the levels after a 72 h incubation in serum-free media are approximately 12.2 +/- 4.4 ng, 272.2 +/- 33.0 ng, and 475.6 +/- 4.8 ng per 10(6) cells per ml (mean +/- SD, n = 3), respectively. In Western blot analyses, a specific monoclonal antibody to tissue kallikrein (V4D11) identifies GH3-secreted kallikrein as a approximately 39,000 Da protein, slightly larger than approximately 38,000 Da kallikreins of submandibular gland, mouse anterior pituitary cells (AtT 20) or rodent neuroblastoma X glioma hybrid cells (NG108). Kallikrein mRNA in GH3 cells was identified in Northern blot analyses, using a tissue kallikrein cDNA probe. In a RIA using a kallikrein monoclonal antibody (V1C3) recognizing only active kallikrein, kallikrein could not be detected in the media incubated up to 48 h with GH3 cells. However, after trypsin treatment, a time-dependent increase of immunoreactive kallikrein (using monoclonal antibody V1C3), Tos-Arg-OMe esterase, and kinin-releasing activities can be measured in the conditioned media. The activated esterase activity was inhibited by aprotinin and by affinity-purified kallikrein monoclonal antibody (V4D11) in a dose-dependent manner. The data indicated that rat anterior pituitary GH3 cells secrete latent tissue kallikrein, which can be converted to active kallikrein by trypsin. These hormonally responsive cells co-synthesize kallikrein with prolactin and growth hormone and provide a model system for studying the regulation of kallikrein gene expression.
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PMID:Identification of latent tissue kallikrein, prolactin and growth hormone secretion in GH3 pituitary cells using modified radioimmunoassays. 336 Feb 6

Glandular kallikrein (a trypsin-like serine protease) is an estrogen-induced and dopamine-repressed protein in the rat anterior pituitary which appears to be associated with lactotrophs. This study examined glandular kallikrein levels in diethylstilbestrol (DES)-induced pituitary tumors in F344 rats and compared it to plasma and pituitary prolactin, and pituitary wet weight. Ovariectomized F344 rats were implanted with Silastic tubes containing 0 or 5 mg DES for 1, 3, 5, 7, or 9 weeks. Glandular kallikrein was measured by microenzymatic assay using D-valylleucylarginyl-p-nitroanilide following trypsin treatment of extracts to activate latent forms of glandular kallikrein. Prolactin was measured by radioimmunoassay. DES induced steady time-dependent increases in pituitary wet weight with 7- and 16-fold increases observed by 5 and 9 weeks, respectively. Growth rates averaged 11.4 mg/week during the first 5 weeks of DES exposure, and then increased to 23.2 mg/week between weeks 5 and 9. Glandular kallikrein total activity (nmol/min/pituitary) increased 130- and 240-fold after 3 and 5 weeks of DES exposure, respectively, and then abruptly plateaued. The specific activity (nmol/min/mg protein) of glandular kallikrein peaked at 3-5 weeks (36-fold increase compared to controls) and then declined as pituitary protein but not glandular kallikrein continued to increase. Total pituitary prolactin constantly rose during DES exposure with 12- and 26-fold increases after 5 and 9 weeks, respectively. Plasma prolactin levels also continuously rose during exposure to DES with 130- and 290-fold increases after 5 and 9 weeks, respectively. No major strain differences were found with regard to sensitivity to the acute effects of estrogen or dopaminergic stimulation on glandular kallikrein induction. DES-induced pituitary tumors in F344 rats are well known to arise via lactotroph proliferation, and the striking elevation in glandular kallikrein and prolactin during the early phases of tumor growth provide further support for a localization of glandular kallikrein in lactotrophs. However, the abrupt stabilization in glandular kallikrein levels by week 5 was unexpected and may signal a biochemical transformation of the tissue during tumor progression.
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PMID:Glandular kallikrein in estrogen-induced pituitary tumors: time course of induction and correlation with prolactin. 339 Aug 8

A prolactin (PRL) inhibitory substance(s) present in rat serum has previously been shown to inhibit the release of PRL from rat pituitary cells in vitro (Hymer and Signorella, 1981). In this study, addition of rat serum to rat pituitary cells cultured in serum-free medium inhibited the release of PRL in dose-dependent manner. Neither the form of the released PRL molecule, nor its biological or immunological activity, was altered by rat serum treatment. Serum prepared from clotted blood previously incubated at 37 degrees C always contained more PRL inhibitory activity than serum from blood incubated at 4 degrees C; whole blood serum, but not plasma-derived serum, contained inhibitory activity. The inhibitor therefore comes from a formed element in the blood. Results of several experiments indicated that buffy coat cells, adherent blood cells and peritoneal cells released the inhibitor, but platelets and RBCs did not; it is suggested that a macrophage/monocyte is probably the cellular source of the PRL inhibitor. Gel permeation high-performance liquid chromatography (HPLC) of rat serum indicated that the inhibitory activity was associated with high molecular weight material, i.e. 115,000-215,000. The PRL inhibitory substance was trypsin sensitive, not extractable with butanol/diisopropyl ether, precipitated at 40% saturated ammonium sulfate, associated with material having an isoelectric point of 4.96 +/- 0.26, and not adsorbed with protein-A. These characteristics suggest the proteinaceous nature of the inhibitory substance. The inhibitor did not suppress PRL release until 9 h after addition to cultured cells, but decreased PRL synthesis was evident within 4 h of treatment. The inability of the dopamine antagonist, haloperidol, to block the inhibitory effect of rat serum indicated that the material does not exert its effect through the dopamine receptor.
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PMID:Characterization of a substance in rat serum which modulates prolactin release in vitro. 378 Oct 96

Studies were carried out in order to characterize specific insulin binding sites in the rat pituitary gland. Binding of labeled insulin by pituitary microsomes reached equilibrium after 4 h at 4 degrees C and remained stable over 16 h; at 25 degrees C the plateau was reached in 20 min. Equilibrium binding data analysis of competitive displacement of bound 125I-iodo insulin by unlabeled insulin yielded a non-linear Scatchard plot. At 25 degrees C the Kd for the high affinity component was 2.8 +/- 0.1 X 10(-9) M and the receptor concentration was 260 +/- 80 fmol/mg of microsomal protein. A Kd value of 4.6 +/- 0.4 X 10(-8) M and a binding capacity of 800 +/- 200 fmol/mg microsomal protein were obtained for the low affinity sites. Insulin binding to microsomes was enhanced 2.7 times by increasing the ionic strength of the incubation medium with 2 M NaCl, and was abolished when the microsome preparation was preincubated with trypsin prior to binding measurements. Other hormones, such as bovine thyrotropin, ovine follitropin, human somatotropin and ovine prolactin did not interact with the insulin receptor. Proinsulin displaced the labeled hormone in direct proportion to its insulin-like biological activity.
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PMID:Specific binding sites for insulin in the rat pituitary gland. 390 55


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